| Objective:With today's technology to improve the means of diagnosis and treatment,many patients with lung cancer can achieve early detection and early treatment of patients with lung cancer,is to bring the gospel,but with the tumor recurrence and metastasis,the death toll is still high.Although the primary tumor can be cured by surgical resection and adjuvant chemotherapy,the recurrence and metastasis of the tumor is still a major problem.At present,according to the theory of CSC?cancer stem cells?proposed by the majority of scholars,CSC is considered to be the basis of tumor proliferation and an important cause of tumor recurrence and metastasis.It has been proved that there are cancer stem cells in many kinds of tumors,but the mechanism of the regulation is still limited.If we can explain and clarify the regulatory mechanism of tumor stem cells,it may lead to a revolutionary breakthrough in the future treatment of cancer.5-?5-fluorouracil,5-Fu?is the basic drug for chemotherapy of malignant tumor,which is widely used in clinical treatment.It belongs to the cell cycle specific drugs for malignant tumors in clinical,mainly in the S phase,many of the chemotherapy regimens are containing fluorouracil drugs.Our team found that 5-Fu enriched cancer cells that were resistant to chemotherapy had many characteristics of cancer stem cells.The study on human lung adenocarcinoma cell line A549 as the experimental object,the application of serum free culture and the role of 5-Fu enrichment of cancer stem cells,by observing the dynamic changes of the expression of stem cell related cytokines Oct3/4,Fbxw7 and Skp2 to explore the mechanism of how to control the import and G0/G1 stage of cancer stem cells.Furthermore,the effects of Fbxw7 and Skp2 on the regulation of c-Myc and P27 were observed.To further investigate the regulatory mechanism of Fbxw7 and Skp2 gene on the A549 cancer stem cells from G0/G1 phase to S phase transformation,namely,the dormancy and activation of A549 cancer stem cells.Materials and Methods:1.Immunohistochemical staining was used to detect the expression of Fbxw7 and Skp2 in lung adenocarcinoma.2.A549 cells were cultured in10%Fetal bovine serum of RPMI-1640,100U/ml penicillin,100U/ml streptomycin,37?,5%CO2 cell culture box.3.MTT determination of the inhibition rate of 5-Fu on the proliferation of A549 cells.4.According to the effect of 5-Fu?IC50?on A549 cells,the A549 cell model of 5-Fu cells was established by 0h and 48h before and after treatment.5.Immunofluorescence staining was used to detect the expression of Fbxw7,c-Myc,Skp2,P27,and Oct3/4 in the A549 cell model of 5-Fu cells.6.A549 cells were cultured in serum-free medium to collect suspended A549 cancer stem cells.7.The siRNA interference A549 cell lines with Fbxw7 and Skp2 were respectively established by transient transfection.8.Detection of Fbxw7,c-Myc,Skp2,P27 and Oct3/4 gene protein expression by Western blot.9.Detection mRNA of Fbxw7,c-Myc,Skp2,P27 and Oct3/4 gene expression in by Real-time PCR.10.Cell cycle was detected by flow cytometry.11.Lung adenocarcinoma xenograft models were employed to detect the effects of Fbxw7 on tumor growth.12.All data used 3 independent experimental results,with mean+standard deviation?mean+SD?to express.SPSS statistical analysis software was used for statistical analysis.T test was used for comparison between groups,P<0.05 was statistically significant.Results:1.The expression of Fbxw7 in lung adenocarcinoma was negative and positive in the adjacent tissues,and the expression was positively correlated with the degree of cell differentiation.The positive expression of Skp2 in lung adenocarcinoma was negative in the adjacent tissues,and the expression was negatively correlated with the degree of cell differentiation.2.5-Fu could inhibit the proliferation of lung cancer A549 cells,and the inhibition rate of cell proliferation was detected by MTT method.It was found that the inhibition was positively correlated with drug concentration and time.According to the results of MTT test,the experiment finally selected 200 g/ml,the role of as a 5-Fu for half of the inhibition of A549 cells?IC50?for the experiment.3.Serum free culture and 5-Fu can enrich the cancer stem cells of A549 cells.The collected suspended stem cells cultured in serum,and the 5-Fu?IC50?function of A549cells before and after stem cell related factor Oct3/4 detection,5-Fu?IC50?before and in serum-free medium before Oct3/4 basically no expression after 5-Fu and serum-free medium after the expression of Oct3/4 positive cells increased significantly.4.Immunofluorescence double staining was used to detect Fbxw7,c-Myc,Skp2,P27 and Oct3/4 before and after the action of 5-Fu?IC50?.It was found that after the action of5-Fu?IC50?,when the expression of Oct3/4 increased,the expression of Fbxw7 and P27increased,and the expression of c-Myc and Skp2 decreased.5.Fbxw7,c-Myc,Skp2 and P27 are related to the regulation of A549 cell cycle.The model of A549 cell attack recovery was established by using 5-Fu?IC50?before,after and after treatment for 0h and 48h.The results showed that the expression of Fbxw7 and c-Myc was reverse,and that of Skp2 and P27 was reverse.The results of cell cycle test showed that the proportion of cells in G0/G1 phase increased significantly after 5-Fu?IC50?was restored to 0h,compared with that before 5-Fu?IC50?,while the proportion of cells in S phase decreased;compared with the group restored to 48h,the proportion of cells in G0/G1phase decreased significantly and the proportion of cells in S phase increased significantly in the group restored to 0h.It can be concluded that Fbxw7,c-Myc,Skp2and P27 are involved in and regulate the dormancy and activation of cancer stem cells in A549 cell line.6.After transient silencing of Fbxw7 and Skp2 by SiRNA interference technology,it was found that Fbxw7 could stabilize A549 cancer stem cells in G0/G1phase by negatively regulating the expression of c-Myc.Skp2 regulates the expression of P27 in a negative direction,which promotes A549 cancer cells to enter S phase.This result has also been verified in animal experiments.Conclusion:1.The expression of Fbxw7 was positively correlated with the degree of cell differentiation.The expression of Skp2 was negatively correlated with the degree of cell differentiation.2.By negatively regulating the expression of c-Myc,Fbxw7 can prevent the transformation of A549cancer stem cells from dormancy to proliferation,that is to say,Fbxw7 can stabilize the dormancy of cancer stem cells.Skp2 can activate cancer stem cells by negatively regulating P27 expression.3.The inhibition of Fbxw7 can push the cancer stem cells into the proliferation stage first,which makes them more easily to be killed by anticancer drugs,and provides a new way to reduce and kill cancer stem cells.4.Inhibition of Skp2can inhibit the activation of cancer stem cells,effectively inhibit the proliferation and division of tumor cells,and provide a basic theoretical basis for long-term maintenance of human cancer coexistence and prolonging the life span of patients. |
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